Method for producing xanthylic acid by microorganisms



United States Patent M 3 211 629 METHOD FOR PROD UCiNG XANTHYLIC ACID BY MICROORGANISMS Shigeo Abe, Tokyo, Kiyoshi Udagawa, Yokohama,

3,211,629 Patented Oct. 12, 1965 imurn nutrient medium (containing each 10 ,LLg./H1l. of amino acids and vitamins) biochemical mutants of each individual bacterium shown in the specific examples of the present application which has the capability of pro- Takashi Nara, Tokyo, and Masanam Misawa Kawa, ducing xanthylic acid requires guanine or its substituted sakihi, Ja an, assignors t Kyowa H kk Kogyo C derivatives or other substances containing guanine Ltd., Tokyo, Japan,acorporation of Japan for its growth in the minimum medium. A culture No h g- Fiied 1963, sen 314,520 medium containing suitable amounts of sugar, other Claims P y, application Japan, 1962, carbon sources, nitrogen sources, inorganic substances, 5 z fiii guanine sources and other nutrients is useful. Guanine sources are free guanine, its derivatives (ribo-, deoxy- The present invention relates to a method for proiiboside TibO', deoxyrihetide), nucleic acids ducing xanthylic acid with the aid of microorganisms. and Other materials (Y eXtfaet, C011- More particularly this invention relates to an economitainihg nucieic acids a their hydroiylates- AS cal method for producing xanthylic acid (xanthosine-5- 15 for microorganisms to be used, bacteria, moulds, Yeasts phosphate) by culturing nutrient-requiring microorgam and other microorganisms which need guanine or its subisms in culture m di t i i inexpensive carbon stituted derivatives or other substances containing guanine sources, nitrogen sources, inorganic substances and nufor their growth can be used- Fermentation is Performed tri nt necessary f th microorganisms under aerobic conditions such as shaking culture or aeroxanthylic acid is a kind f Purine nucleotide and an bic stirring culture; the culturing temperature is from 2.0 important intermediate in the biosynthesis of ribonucleic t0 and the P 0f the culturing medium is p acid and deoxyribonucleic acid in cells of microorgaat t0 If necessary, a neutralizing agent is usednisms. As is well known, xanthylic acid has a widespread After a culturing Period of 2 to 8 y bacteria and use in the field of food seasonings, medicines and the like. yeasts, 3 to 10 y for moulds and -P myce a con- A primary bj t of th present invention i h siderable amount of xanthylic acid is accumulated in the bodiment of an economical method for roducing nucleic fermentation medium and the cells Of microorganisms. acid constiuting components and metabolic components After the fermentation is Over, the resultant Product is with the aid of microorganisms and is based on the ob- Separated servation that certain kinds of nutrient-requiring micron r the method of this invention lies in h use of organisms (biochemical mutants) accumulate a large biochemical mutants which are Produced y inducing amount of Xanthylic acid in fermentation medium. As mutation of known microorganisms y P 56 known hereinafter described, the objective commercially feasible methods, effecting direet fermentation With Said mutants production method has been embodied by providing suitin the Culture medium containing Cheap Sugar Sources, or able conditions of culture for the accumulation of xanthy- Carbon Sources, nitrogen Sources, inorganic Substances, lic acid, By employing Micrococ u lm i 534 guanine sources and other nutrients, thereby accumulat- (ATCC 13032) and Br ib z ri ammgn 'agenes ing xanthylic acid in the fermentation medium, and ob- (ATCC 6871 and 6872) as individual original strains, taining the product after separating either by single or repeated treatment thereof by irradiation (UV-ray, 'yby simultaneous use of ion-exchange treatment, adsorpray, etc.) or by chemical agents (nitrogen mustard, etc) tion method, precipitation method and extraction method.

Strain used Original strain Strain and No. Requiring Strain and N0. Requiring nutrient nutrient Micrococcus glutamz'cus Guanine and Micrococcus glutamicus Biotin.

No. 02101 (ATCC 15135). biotin. 534 (ATCC 13032). Micrococcus glutamicus Do. Micrococcus glutamicus Do.

No. 62110 (ATCC 15136). 534 (ATCC 13032). Brevibacterium ammoniagenes D0. Brevibacterium ammoni- Do.

No. 62201 (ATCC 15137). agenes (ATCC 6871). Brevz'bacterium ammoniagenes Do. Brevibacterium ammoni- Do.

No. 62221 (ATCC 15138). agenes (ATCC 6872). Brevibacterium ammoniagenes D0. Brem'bacierium ammoni- Do.

No. 62223 (ATCC 15139). agenes (ATCC 6872).

The following examples of presently-preferred embodiments of the present invention are for the purpose of illustration only and are not intended to be restrictive. Percentages are by weight per volume.

Example 1 A mutant strain of Micrococcus glutamicus No. 62101 (ATCC No. 15135) (guanine and biotin requiring strain) is used. The strain is inoculated in the following preculture medium and subjected to shaking culture at 28 C. for 24 hours. The seed preculture medium is com posed of 2 percent of glucose, 1 percent of peptone, 0.5 percent of yeast extract and 0.2 percent of sodium chloride, and pH of the medium is adjusted to 7.0. A 10 milliliter (ml.) aliquot of the medium is placed in a large test tube and sterilized at 120 C. for 10 minutes. Seed culture broth is inoculated into the following fermentation medium and cultured for 72 hours.

The fermentation medium is composed of 10 percent of glucose, 0.5 percent of KH PO 0.5 percent of K HPO 0.3 percent of urea, 0.2 percent of (NH SO 0.03 percent of MgSO -7H O, 0.01 percent of peptone, 0.01 percent of yeast extract, 0.1 percent of corn steep liquor, 10 g/ml. of guanine, and 2 percent of CaCO A 30 ml. aliquot of this medium in a 250 m1. conical flask, after being adjusted to pH 7.2, is sterilized at 120 C. for 10 minutes.

5.6 mg./ml. of xanthylic acid accumulates in the fermentation broth. From this broth, the microorganism cells and insoluble CaCO are removed. 1 liter of the resultant filtrate is made to pH 0.8 with 1 N-HCl and passed through a strongly acidic sulfonic acid-type cation exchange resin (Diaion SK No. 1,-H type, product of Mitsubishi Chemicals Co., Ltd.) to adsorb xanthylic acid. After passing the filtrate, the resin is Washed by water. The adsorbed xanthylic acid is eluted with HCl. Effluent containing xanthylic acid and the first portion of washing liquor are combined and concentrated under vacuum at 30 C. after being adjusted to pH 7.2 with saturated Ba(OH) When 200 ml. of ethanol are added to 100 ml. of concentrated liquor and allowed to stand still for one whole day, crystals of barium salt of xanthylic acid are formed. After filtration and collection, the barium salt is converted to sodium salt by treatment with Na SO By elementary analysis, analysis of ribose, xanthine and phosphoric acid content, absorption spectrum of ultraviolet ray and infra-red ray, the resulting product is proved to be sodium salt of 5'-xanthylic acid. The yield is 4.1 g.

No accumulation of xanthylic acid with Micrococcus glutamicus 534 (ATCC 13032), original strain, is observed.

Example 2 A mutant strain of Brevibacterium ammonz'agenes No. 62201 (ATCC No. 15137) (guanine and biotin requiring strain) is used. Fermentation medium is composed of 8 percent of glucose, 0.01 percent of casein hydrolyzate, 0.7 percent of yeast extract, 1.0 percent of (NH SO 0.3 percent of KH PO 0.3 percent of K HPO 0.05 percent of MgSO -7H O, 10 ,ug./ml. of guanine, and 10 ug/liter of biotin. It is adjusted to pH 7.2 before sterilization and 2 percent of sterilized CaCO is added after sterilization. ple 1. By culture for 76 hours, 4.8 mg./ml. of 5'-xanthylic acid is accumulated.

No accumulation of xanthylic acid with Brevibacterium ammoniagenes ATCC 6871, original strain, is observed.

Example 3 Using a mutant strain of Brevibacterium ammoniagenes No. 62221 (ATCC No. 15138) (guanine and biotin requiring strain) culturing is performed under the same conditions as in Example 2. After culturing for 72 hours, 6.9 mg./ml. of 5'-xanthylic acid has accumulated in the fermentation broth.

Other conditions are the same as in Exam- No accumulation of xanthylic acid with Brevibacterium ammoniagenes ATCC 6872, original strain, is observed.

Example 5 Using a mutant strain of Brevibacterium ammoniagenes No. 62223 (ATCC No. 15139) (guanine and biotin requiring strain), culturing is performed under the same conditions as in Example 2. After 75 hours of fermentation 3.5 mg./ml. of 5'-xanthylic acid has accumulated.

In each of Examples 2, 3, 4 and 5, the accumulated xanthylic acid can be recovered after the manner described in Example 1.

These yields are as follows.

Example: Yield, g. 2 4.1 3 5.5 4 5.9 5 3.0

Example 6 Employing the same mutant strain as in Example 4, culturing is performed under the same medium as in Example 2, excepting that no guanine is added and the amount of yeast extract is 1 percent. After 73 hours of fermentation 5.4 mg./ml. of 5-xanthylic acid has accumulated.

The yield recovered after the manner described in Example 1 is 4.8 g.

Example 7 Example 6 is repeated in the same medium as in Example 2, excepting that the amount of guanine is 20 g./ ml. and no yeast extract is added. After 73 hours of fermentation 4.9 mg./ml. of 5-xanthylic acid has accumulated.

The yield recovered after the manner described in Example 1 is 4.2 g.

Example 8 Example 6 is repeated with the same medium as in Example 2, excepting that guanine and yeast extract are not added, and guanosine is added in the amount of 35 g/ ml. After 74 hours of fermentation 4.5 mg./ml. of 5'- xanthylic acid has accumulated.

The yield recovered after the manner described in Example 1 is 4.0 g.

What is claimed is:

1. A method which comprises (a) aerobically culturing Micrococcus glutamicus No. 62101 (ATCC 15135) under fermentation conditions in a fermentation medium containing carbon sources, nitrogen sources, inorganic substances, guanine sources, and biotin, whereby 5-xanthylic acid is directly produced and accumulated, (b) isolating and (c) recovering from the medium the substance so produced and accumulated.

2. A method which comprises (a) aerobically culturing Micrococcus glutamicus No. 62110 (ATCC 15136) under fermentation conditions in a fermentation medium containing carbon sources, nitrogen sources, inorganic substances, guanine sources, and biotin, whereby 5-xanthylic acid is directly produced and accumulated, (b) isolating and (c) recovering from the medium the substance so produced and accumulated.

3. A method which comprises (a) aerobically culturing Brevibacterium ammoniagenes No. 62201 (ATCC 15137) under fermentation conditions in a fermentation medium containing carbon sources, nitrogen sources, inorganic substances, guanine sources and biotin, whereby 5'-xanthylic acid is directly produced and accumulated, (b) isolating and (c) recovering from the medium the substance so produced and accumulated.

4. A method which comprises (a) aerobically culturing Brevibacterium ammoniagenes No. 62221 (ATCC 15138) under fermentation conditions in a fermentation medium containing carbon sources, nitrogen sources, inorganic substances, guanine sources and biotin, whereby 5'-Xanthylic acid is directly produced and accumulated, (b) isolating and (c) recovering from the medium the substance so produced and accumulated.

5. A method which comprises (a) aerobically culturing Brevibacterium ammoniagenes No. 62223 (ATCC 15139) under fermentation conditions in fermentation medium containing carbon sources, nitrogen sources, in-

5-Xanthylic acid is directly produced and accumulated, (b) isolating and (c) recovering from the medium the substance so produced and accumulated.

References Cited by the Examiner UNITED STATES PATENTS 3,102,079 8/63 Kinoshita. 3,152,966 10/64 Kinoshita et al.

organic substances, guanine sources and biotin, whereby 10 A. LOUIS MONACELL, Primary Examiner. 

1. A METHOD WHICH COMPRISES (A) AEROBICALLY CULTURING MICROCOCCUS GLUTAMICUS NO. 62101 (ATCC 15135) UNDER FERMENTATION CONDITIONS IN A FERMENTATION MEDIUM CONTAINING CARBON SOURCES, NITROGEN SOURCES, INORGANIC SUBSTANCES, GUANINE SOURCES, AND BIOTIN, WHEREBY 5''-XANTHYLIC ACID IS DIRECTLY PRODUCED AND ACCUMULATED, (B) ISOLATING AND (C) RECOVERING FROM THE MEDIUM THE SUBSTANCE SO PRODUCED AND ACCUMULATED.
 3. A METHOD WHICH COMPRISES (A) AEROBICALLY CULTURING BREVIBACTERIUM AMMONIAGENES NO. 62201 (ATCC 15137) UNDER FERMENTATION CONDITIONS IN A FERMENTATION MEDIUM CONTAINING CARBON SUCES, NITROGEN SOURCES, INORGANIC SUBSTANCES, GUANINE SOURCES AND BIOTIN, WHEREBY 5''-XANTHYLIC ACID IS DIRECTLY PRODUCED AND ACCUMULATED, (B) ISOLATIONG AND (C) RECOVERING FROM THE MEDIUM THE SUBSTANCE SO PRODUCED AND ACCUMULATED. 